Mitochondrial Ca2+ handling is central to cellular calcium homeostasis, energy metabolism and cell survival/cell death mechanisms. Impaired mitochondrial Ca2+ transport is also thought to be an important contributor in the development of a variety of diseases. After decades of frustrating ambiguity about the molecules mediating mitochondrial Ca2+ uptake, in 2010-2011, MICU1, an EF-hand domain protein, and MCU were identified as an indispensible regulator and pore forming component of the Ca2+ uniporter, respectively. Now, it is becoming possible to tackle the fundamental questions about the molecular mechanisms that underlie a sophisticated Ca2+ transport system and the physiological significance of MICU1, MCU and other uniporter components. We have started to investigate the mode of action of MICU1, and contrary to the first report, we found that MICU1 is not required to allow Ca2+ uptake through the uniporter but, instead, is critical to keep the uniporter closed at low cytoplasmic [Ca2+] ([Ca2+]c) concentrations and to support cooperative activation of the uniporter as the [Ca2+]c increases. These studies have been recently published in a widely recognized paper in Cell Metabolism. To further the study of the physiological significance of MICU1 we have generated a conditional MICU1 knockout mouse that has a striking phenotype and will provide several models for the proposed study. Our hypotheses are that (1) MICU1 employs its C-terminal cluster of positively charged amino acid residues to interact with the DIME domain of the intermembrane space loop of MCU, highlighting a site that might be a novel drug target, (2) MICU1 and MICU2 differently bind Ca2+ and/or Mg2+ with their EF-hands, providing a possible mechanisms for their distinct effects on the cooperative activation of the MCU, and (3) MICU1 is required in vivo for effective decoding of calcium signals in terms of oxidative metabolism in hepatocytes under fed and fasting conditions. To explore the role of MICU1 in Ca2+ handling we will apply advanced fluorescence imaging approaches, including some novel methods combined with assessment of cell bioenergetics and the activity of cell survival regulating pathways in the same models. The proposal relies on the use of a powerful targeting system of MICU1 in both cell lines and mouse tissues. The proposed work will uncover the mechanism of the sophisticated Ca2+- and time-dependent control of the uniporter and will expose MICU1's role in keeping mitochondria healthy and responsive to Ca2+.